Surgical resection, chemotherapy, radiotherapy and immunotherapy have been mainly used alone or together as a method for treating cancer (malignant tumor). Among them, the immunotherapy has a lot of potentialities and is therefore expected to make further progress in the near future although it is still in a developmental stage now.
The cancer-specific immunotherapy the means a treating method in which a cytotoxic activity is affected only upon cancer cells. As a drug showing the cytotoxic activity is combined with an antibody so as to have directivity in this therapy, it is now called a “missile therapy.” Studies have now been carried out by targeting a substance that is abnormally expressed in the cancer cells or that will change according to malignant alternation or canceration of cells and using said substance as an antigen that will be effectively used for the preparation of the antibody with a minimum of side effects. Such antigen is called a cancer-associated antigen.
Among antibodies with multiple specificities, an antibody with bispecificity (Bispecific Antibody: BsAb) has been studied intensively. The bispecific antibody can bind specifically to two different kinds of antigens so that it will be utilized as a therapeutic agent having a specific anti-cancer effect. A diabody (Db) is a minimum unit of the above bispecific antibody. It was developed by utilizing the property that the variable region in a heavy chain (VH) and the variable region in a light chain (VL) derived from the same parent antibody will form a hetero-dimer through non-covalent bond (Hollinger, et al., Proc. Natl. Acad. Sci. USA 90, 6444-6448, 1993).
The diabody-type bispecific antibody is characterized by having low immunogenicity and high infiltrating activity into tumor tissues due to its low molecular weight (ca. 60,000), and by being able to be easily mass-produced at a low cost with use of microorganisms such as E. coli, and to be easily altered in function by means of genetic engineering.
The present inventors already found that the diabody-type bispecific antibody (Ex3), which was produced by utilizing an anti-human EGF receptor 1 (Her 1) antibody 528 and an anti-CD3 antibody OKT3, and its humanized diabody-type bispecific antibody (referred to as “hEx3” in Patent Document 1) showed an extremely strong anti-tumor effects. It was further speculated that the structural stability of the variable regions of the above antibodies 528 and OKT3 themselves and their combination are very important for showing such advantageous effects by comparison with an diabody-type bispecific antibody prepared using other antibodies.
Methods for the production of bispecific antibodies other than the diabody-type bispecific antibody are described in Non-Patent Documents 1 and 2.
Patent Document 1: Japanese Patent Publication No. 2004-242638
Non-Patent Document 1: Alt M, et. al. Novel tetravalent and bispecific IgG-like antibody molecules combining single-chain diabodies with the immunoglobulin gamma1 Fc or CH3 region. FEBS Lett., 454, 90-4. (1999)
Non-Patent Document 2: Lu D, et. al. A fully human recombinant IgG-like bispecific antibody to both the epidermal growth factor receptor and the insulin-like growth factor receptor for enhanced antitumor activity. J Biol. Chem., 280, 19665-72. (2005)